National Institute on Alcohol Abuse and Alcoholism National Institutes of Health

Bin Gao, MD PhD Chief, Laboratory of Liver Diseases

(45 min)

Alcoholic Liver Disease:

Pathophysiology, Treatment, and Challenges

Alcohol

Fatty Liver

Fatty Liver

60-70%

Alcoholic Steatohepatitis (ASH)

Inflammation 30-40%

Cirrhosis

10-20%

1-3% per year

Gao and Bataller, Gastroenterology 2011, 141(5):1572-85

Acute (sub-acute) Alcoholic Hepatitis Clinical syndrome: Jaundice, hepatomegaly, fever, Neutrophilia, AST: ALT ratio>2

Alcoholic Liver Disease

Alcohol

Fatty Liver

Fatty Liver

60-70%

Alcoholic Steatohepatitis (ASH)

Inflammation 30-40%

Fibrosis Cirrhosis

10-20%

1-3% per year

Gao and Bataller, Gastroenterology 2011, 141(5):1572-85

How is ethanol metabolized in the liver?

ADH: Alcohol Dehydrogenase; ALDH2: Aldehyde Dehydrogenase 2; CYP2E1: cytochrome P450 2E1; NAD+: oxidized nicotinamide adenine dinucleotide; NADH: reduced nicotinamide adenine dinucleotide; NADP+: oxidized nicotinamide adenine dinucleotide phosphate; NADPH: reduced nicotinamide adenine dinucleotide phosphate

Ethanol Acetaldehyde ADH

Zakhari and Li: Hepatology 2008 Hepatocytes

ALDH2: Aldehyde Dehydrogenase 2 polymorphism

Glu504 homozygotes (ALDH2*1/1) exhibit normal enzymatic activity to metabolize acetaldehyde

Glu504/Lys504 heterozygotes (ALDH2*1/2) show approximately 6% of normal activity,

Lys504 homozygotes (ALDH2*2/2) show negligible activity toward acetaldehyde

40-5

0%

East

Asi

ans

5-8%

Ea

st A

sian

s

Acetaldehyde Acetate

Acetaldehyde

Flushing

Acetate

Glu504 Glu504

Glu504 Lys504

Lys504 Lys504

ALDH2: Aldehyde Dehydrogenase 2 polymorphism

40-5

0%

East

Asi

ans

5-8%

Ea

st A

sian

s

Alcoholic liver disease in patients with ALDH2 deficiency has not been characterized, which may be very different from the patients with ALDH2*1/1

One of major projects in our lab at NIAAA The 1st International symposium in Shanghai in 2016

Alcoholic liver disease in patients with this genotype has been extensively characterized in Western countries.

(Ethanol)

(Acetaldehyde)

(ADH1)

(ALDH2)

(Acetate)

30-50% East Asians Asian Americans

ADH1B ADH1c

ALDH2*2 (50%)

High activity

Low activity

Alcohol flush Reaction 脸红

Ethanol metabolism in East Asians , Asian Americans

Are individuals who have alcohol flushing reaction more susceptible to alcoholic liver injury and inflammation?

Kwon HJ1, Won YS, Park O, Chang B, Duryee MJ, Thiele G, Matsumoto A, Singh S, Abdelmegeed M, Song B, Kawamoto T, Vasiliou V, Thiele G, Gao B. .Hepatology 2014 Jul;60(1):146-57.

ALDH2 knockout mice

Ethanol Acetaldehyde

Fatty liver and serum ALT

Liver fibrosis and inflammation Flushing in mice?

Ethanol People with ALDH2 deficiency (Flushing)

Acetaldehyde Fatty liver and serum ALT Liver fibrosis and inflammation

? ?

Alcohol

Fatty Liver

Fatty Liver

60-70%

Alcoholic Steatohepatitis (ASH)

Inflammation 30-40%

Fibrosis Cirrhosis

10-20%

1-3% per year

Gao and Bataller, Gastroenterology 2011, 141(5):1572-85

How is ethanol metabolized in the liver?

How does ethanol cause liver injury?

Ethanol

Molecular Mechanisms of Alcoholic Fatty Liver

a. Ethanol metabolites do not contribute directly to fat synthesis

Gao and Bataller. Alcoholic liver disease: Pathogenesis and therapeutic targets. Gastroenterology 2011, 141:1572-85 Purohit, Gao, Song. Molecular mechanisms of alcoholic fatty liver. Alcohol Clin Exp Res. 2009, 33:191-205

b. Ethanol leads to fatty liver via dysregulating fat metabolism

Alcohol, its metabolites, its metabolism-associated oxidative stress promote fat synthesis and inhibit fatty acid β oxidation

Molecular Mechanisms of Alcoholic Steatohepatitis

Gut bacterial Dysbiosis

Gut permeability Oxidative stress

Lipid peroxidation

Inflammation

Alcohol

Endotoxin Cytokines Oxidative

stress

Gao and Bataller. Alcoholic liver disease: Pathogenesis and therapeutic targets. Gastroenterology 2011, 141:1572-85

Molecular Mechanisms of Alcoholic Cirrhosis

Gao and Bataller. Alcoholic liver disease: Pathogenesis and therapeutic targets. Gastroenterology 2011, 141:1572-85

Gao and Bataller. Alcoholic liver disease: Pathogenesis and therapeutic targets. Gastroenterology 2011, 141:1572-85

Cirrhosis is a slowly progressing disease in which healthy liver tissue is replaced with scar tissue that is mainly produced by activated hepatic stellate cells

Alcohol Acetaldehyde

Oxidative stress Cytokines

Alcohol

Stellate cell activation

Alcohol

Fatty Liver

Fatty Liver

60-70%

Alcoholic Steatohepatitis (ASH)

Inflammation 30-40%

Fibrosis Cirrhosis

10-20%

1-3% per year

Gao and Bataller, Gastroenterology 2011, 141(5):1572-85

How is ethanol metabolized in the liver?

How does ethanol cause liver injury?

Ethanol Only 30-40% of heavy drinkers develop more severe forms of ALD, such as advanced ASH,

fibrosis and cirrhosis.

Only 30-40% of heavy drinkers develop more severe forms of ALD, such as advanced ASH, fibrosis and cirrhosis.

Gao and Bataller. Alcoholic liver disease: Pathogenesis and therapeutic targets. Gastroenterology 2011, 141:1572-85

Many genetic and acquired factors are involved: • Nutritional factors • The dose and duration of alcohol consumption •  Drinking Pattern • Age of onset of drinking • Genetic polymorphisms of cytokines and alcohol-metabolizing enzymes • Gender • Immunologic factors • Genetic predisposition to alcohol addiction • Hepatic iron overload • Comorbidity (viral hepatitis, obesity, diabetes etc.)

Gao and Bataller. Alcoholic liver disease: Pathogenesis and therapeutic targets. Gastroenterology 2011, 141:1572-85

Many genetic and acquired factors are involved: • Nutritional factors • The dose and duration of alcohol consumption •  Drinking Pattern • Age of onset of drinking • Genetic polymorphisms of cytokines and alcohol-metabolizing enzymes • Gender • Immunologic factors • Genetic predisposition to alcohol addiction • Hepatic iron overload • Comorbidity (viral hepatitis, obesity, diabetes etc.)

Only 30-40% of heavy drinkers develop more severe forms of ALD, such as advanced ASH, fibrosis and cirrhosis.

a b c a b

Binge Chronic feeding + Alcoholic Hepatitis

Nature Protocols 2013

Adeline Bertola, Stephanie Mathews, Sung Hwan Ki, Hua Wang & Bin Gao

Control Chronic ethanol 8 weeks

ALT

U/L

300

200

100

0

Chronic ethanol 8 weeks + one single binge

Chronic-plus-binge ethanol feeding induces severe liver injury

Alcohol Use and Obesity in the United States More than two-thirds of U.S. adults are overweight or obese Prevalence of Drinking: In 2012, 87.6% of people ages 18 or older reported that they drank alcohol at some point in their lifetime; 71% reported that they drank in the past year; 56.3% reported that they drank in the past month.

Alcohol and Obesity

How does the interaction of alcohol and fat affect the liver?

Alcoholic Steatohepatitis (ASH)

Spectrum of Alcoholic Liver Disease (ALD) and Non-alcoholic Fatty Liver Disease (NAFLD)

Non-alcoholic Steatohepatitis (NASH)

Obesity

Steatosis

3-10% ?

Cirrhosis HCC

?

NASH to HCC without cirrhosis

Alcoholic liver disease (ALD): diagnosis ALD diagnosis is based on: •  A past and present history of significant alcohol

drinking (Man: ≥20-30 g/d; Women: ≥14 g/d ) •  Clinical evidence of liver disease •  Elevation of serum AST and ALT (AST/ALT>2) •  Serum AST is elevated to a level of 2-6 times the

upper limits of normal in severe alcoholic hepatitis. •  AST/ALT ratio > 3 is highly suggestive of ALD

Robert S. O’Shea, Srinivasan Dasarathy, Arthur J. McCullough Alcoholic liver disease (AASLD guideline) Hepatology 2010

Nonalcoholic fatty liver disease (NAFLD): diagnosis

NAFLD diagnosis : •  The diagnosis of NAFLD requires: hepatic

steatosis (exclude other causes of hepatic fat accumulation)

•  In the majority of patients, NAFLD is associated with metabolic risk factors such as obesity, diabetes mellitus, and dyslipidemia.

•  NAFLD is histologically further categorized into Nonalcoholic fatty liver (NAFL) Nonalcoholic steatohepatitis (NASH) •  Elevation of serum AST and ALT (AST/ALT<1)

Alcoholic Steatohepatitis (ASH)

Spectrum of Alcoholic Liver Disease (ALD) and Non-alcoholic Fatty Liver Disease (NAFLD)

Non-alcoholic Steatohepatitis (NASH)

Obesity

Steatosis

3-10%

Cirrhosis HCC

Obesity and alcohol contribute to the pathogenesis of fatty liver disease. How much for each of them?

http://www.mayoclinic.org/medical-professionals/model-end-stage-liver-disease/alcoholic-liver-disease-nonalcoholic-fatty-liver-disease-index

The ANI is a novel scoring system that is highly accurate in distinguishing ALD from NAFLD. The ANI may be a useful tool for the frequent clinical scenarios in which it is useful to ascertain alcoholic liver injury. Short short-term abstinence does not significantly affect the performance characteristics of the ANI therefore the ANI is unlikely to be useful in detecting surreptious alcohol consumption in patients with known ALD. Other liver diseases should first be excluded before utilizing the ANI. The ANI is most accurate when the MELD Score is below 20. Please enter values and press Calculate ANI. AST: IU/I ALT: IU/I MCV: fL Weight: kg/lb Height: m/in Gender: Male Female ANI score: Probability of ALD:

How to distinguish ALD and NAFLD? ANI Mayo clinic: The Alcoholic Liver Disease (ALD) /Nonalcoholic Fatty Liver Disease (NAFLD) Index (ANI)

ASH or NASH AST/ALT ratio >2 AST/ALT ratio <1

AST/ALT ratio >2 AST/ALT ratio <1

Liver mitochondrial injury Hepatocyte injury AST and ALT

Heart and muscle mitochondrial injury AST

ALT and AST Hepatocyte injury

How to distinguish ALD and NAFLD? AST/ALT ratio

Alcoholic Steatohepatitis (ASH)

Spectrum of Alcoholic Liver Disease (ALD) and Non-alcoholic Fatty Liver Disease (NAFLD)

Non-alcoholic Steatohepatitis (NASH)

Obesity

Steatosis

3-10%

Cirrhosis HCC

Excessive alcohol drinking exacerbates nonalcoholic fatty liver disease, cirrhosis, and liver cancer •  Moderate alcohol drinking? •  Endogenous ethanol?

Dunn et al. , Modest alcohol consumption is associated with decreased prevalence of steatohepatitis in patients with NAFLD. J Hepatol. 2012;57:384-91. Dunn et al., Modest wine drinking and decreased prevalence of suspected nonalcoholic fatty liver disease. Hepatology. 2008;47:1947-54.

Zhu et al.: Characterization of gut microbiomes in nonalcoholic steatohepatitis (NASH) patients: a connection between endogenous alcohol and NASH. Hepatology. 2013, 57:601-9.

In NASH patients: gut alcohol-producing bacteria ↑ → blood ethanol ↑ → ethanol metabolism, oxidative stress, liver inflammation ↑ → NSAH

•  Moderate alcohol drinking? •  Endogenous ethanol? More studies?

Alcohol

Fatty Liver

Fatty Liver

60-70%

Alcoholic Steatohepatitis (ASH)

Inflammation 30-40%

Cirrhosis

10-20%

1-3% per year

Gao and Bataller, Gastroenterology 2011, 141(5):1572-85

Acute (sub-acute) Alcoholic Hepatitis Clinical syndrome: Jaundice, hepatomegaly, fever, Neutrophilia, AST: ALT ratio>2

1.  Stop drinking 2.  Supporting treatment 3.  There are no approved anti-fibrotic drugs. 4.  Management of alcoholic cirrhosis and HCC: similar to other causes 5.  Treatment of alcoholic hepatitis (AH): high mortality Steroid (prednisone) , Novel therapeutic targets: interleukin-22

Therapy of Alcoholic Liver Disease (ALD)

Mathurin P, et al. Corticosteroids improve short-term survival in patients with severe alcoholic hepatitis: meta-analysis of individual patient data. Gut 2011;60:255-60. di Mambro AJ, et al. In vitro steroid resistance correlates with outcome in severe alcoholic hepatitis. Hepatology 2011;53:1316-22. Louvet A, et al. Infection in patients with severe alcoholic hepatitis treated with steroids: early response to therapy is the key factor. Gastroenterology 2009;137:541-8. Singal AK, Shah VH. Alcoholic hepatitis: prognostic models and treatment. Gastroenterol Clin North Am 2011;40:611-39. Christensen E. Glucocorticosteroids in acute alcoholic hepatitis: the evidence of a beneficial effect is getting even weaker. J Hepatol 2010;53:390-1.

Over the years the evidence in favour of glucocorticosteroid therapy in alcoholic hepatitis has steadily decreased, and now it seems to be the time to move on to other therapies with more potential, e.g., pentoxifylline.

Controversies in steroid treatment for alcoholic hepatitis Prednisolone for alcoholic hepatitis 1971 2015

NIAAA supports four large consortia to conduct translational research for exploring

novel therapeutic targets for alcoholic hepatitis

Oxidative stress Mitochondrial injury

Hepatocyte death

Gut flora (bacteria) Gut permeability

Oxidative stress Malondialdehyde 4-hydroxynoneal

Acetaldehyde

Neutrophils, NKT Kupffer cells/ Macrophages

LPS, complements, proinflammatory cytokines

(TNF-α, IL-1β, IL-17 etc) Chemokines (IL-8, CXCL1 etc)

Protein adducts (Antigen?) Antigen presenting cells

(DC, macrophages)

Alcohol

Pathogenesis of AH: Inflammation

Liver Injury Impaired Regeneration

Combination therapy: Anti-inflammation + Hepatoprotection

Steroids Interleukin-22

Liver Injury Impaired Regeneration Complications

Bacterial infection Renal injury

Combination therapy: new hope for alcoholic hepatitis (AH)?

Bin Gao, MD PhD, Laboratory of Liver Diseases, NIAAA, NIH, Bethesda, MD, USA

Vijay Shah, MD

Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA

Interleukin-22

Anti-bacteria Renoprotection

Interleukin-22 is probably the only cytokine that is produced by immune cells but does target immune cells due to lack of IL-22 receptor 1 (IL-22R1) on these cells.

Interleukin-22 mainly targets epithelial cells (eg. hepatocytes), hepatic stellate cells, and some fibroblasts because of high levels of IL-22R1 on these cells.

Key points:

Key functions of Interleukin-22 in the liver: Induces hepatocyte survival Induces hepatocyte proliferation Induces expression of anti-bacterial genes

Radaeva et al. Hepatology 2004

Phase I clinical trial in healthy volunteers (finished)(Generon) Phase IB or Phase II for alcoholic hepatitis is under consideration

Challenges for alcoholic liver disease (ALD)

1.  Stop drinking, Overcoming an addiction, 2.  Pathogeneses of ALD remain obscure. 3.  Non-invasive markers for diagnosis of early ALD 4.  There are no approved drugs for the treatment of

ALD. 5.  Animal models of ALD